Seventy million people have pre-diabetes or diabetes and diabetes accounts for 1 in 7 health care dollars. Understanding the pathogenesis of and developing better therapies for these conditions is a very high priority. The deuterated water and isotopomer methods are extensively used to measure gluconeogenesis in humans. Both methods assume negligible transaldolase exchange. Studies performed during the previous grant period have called this assumption into question. Specific Aim I will determine whether transaldolase exchange occurs in humans and whether it is increased in individuals with impaired fasting glucose (IFG) by infusing [1-13C] acetate and determining whether the [3-13C] UDP-glucose/ [4-13C] UDP-glucose ratio is less than 1.0 indicating the presence of transaldolase exchange. Individuals also will be given 2H2O and euglycemic clamps performed so the data from those experiments can be used in Specific Aim II to test the hypotheses that a) people with IFG and normal glucose tolerance (IFG/NGT) have hepatic insulin resistance, b) the severity of hepatic insulin resistance is greater in people with combined IFG/IGT than IFG/NGT, and c) hepatic insulin resistance in IFG results from impaired suppression of glycogenolysis as well as gluconeogenesis. We also established during the previous grant period that the splanchnic bed converts large amounts of cortisone to cortisol and provided evidence that virtually all of this conversion occurs within the liver. This suggests that generation of cortisol via the 11 2-HSD type 1 pathway may be an important regulator of hepatic insulin action. Specific Aim III will develop and use a novel triple tracer approach to determine if obesity or type 2 diabetes decreases the rate of hepatic cortisone to cortisol conversion by having lean non-diabetic, obese non-diabetic and obese diabetic subjects ingest [9,12,12-2H3] cortisol ([2H] cortisol) and [4-13C] cortisone([13C] cortisone). Rates of appearance of [2H] cortisol and [13C] cortisol will be measured using intravenously infused [1,2,6,7-3H] cortisol ([3H] cortisol) in order to concurrently measure both hepatic extraction of the ingested [2H] cortisol and hepatic conversion of the ingested [13C] cortisone to [13C] cortisol thereby enabling measurement of hepatic 11 2-HSD-1 flux without the need for hepatic venous catheterization. Since virtually nothing is known regarding the hormonal regulation of hepatic 112-HSD-1 activity in humans, Specific aim IV will determine whether insulin decreases and glucocorticoids increase hepatic cortisol production by using a randomized study design in which subjects will either ingest hydrocortisone or placebo for one week followed by either a hyperinsulinemic euglycemic clamp or saline infusion combined with the methods outlined in Specific aim III. If so, this would be consistent with observations in vitro and would provide the stimulus for additional studies seeking to determine whether impaired suppression of hepatic 112- HSD-1 by insulin and/or excessive stimulation due to subtle elevations in circulating cortisol contribute to the hepatic insulin resistance in obesity and type 2 diabetes. PUBLIC HEALTH RELEVANCE: Almost 70 million people in the United States have either pre-diabetes or diabetes. These conditions cause a tremendous amount of human suffering and result in enormous costs to the economy as well as the heath care system. The ultimate goal of the studies proposed in this application is to understand the cause of these disorders in order to develop rational approaches for their prevention, treatment and hopefully ultimate cure. In particular, this application will focus on how and why the liver releases too much glucose into the blood stream causing blood sugars to rise both before and after eating.